Earthworms as vectors of Escherichia coli O157:H7 in soil and
vermicomposts
A. Prysor Williams1, Paula Roberts1, Lisa M. Avery2, Ken Killham3 & David L. Jones1
1
School of Agriculture and Forest Sciences, University of Wales, Bangor, Gwynedd, UK; 2School of Water Sciences, Cranfield University, Cranfield,
Bedford, UK; and3Department of Plant and Soil Science, University of Aberdeen, Cruickshank Building, Aberdeen, UK
Correspondence: Prysor Williams, School of
Agricultural and Forest Sciences, University of
Wales, Bangor, Gwynedd, LL57 2UW, UK.
Tel.: 144 1248 382579; fax: 144 1248
354997; e-mail: [email protected]
Received 2 November 2005; revised 31 January
2006; accepted 23 February 2006.
First published online 10 May 2006.
DOI:10.1111/j.1574-6941.2006.00142.x
Editor: Julian Marchesi
Keywords
contamination; earthworms; Escherichia coli
O157:H7; soil; survival; vermicompost.
Abstract
Survival and movement of Escherichia coli O157:H7 in both soil and vermicompost
is of concern with regards to human health. Whilst it is accepted that E. coli
O157:H7 can persist for considerable periods in soils, it is not expected to survive
thermophilic composting processes. However, the natural behavior of earthworms
is increasingly utilized for composting (vermicomposting), and the extent to
which earthworms promote the survival and dispersal of the bacterium within
such systems is unknown. The faecal material produced by earthworms provides a
ready supply of labile organic substrates to surrounding microbes within soil and
compost, thus promoting microbial activity. Earthworms can also cause significant
movement of organisms through the channels they form. Survival and dispersal of
E. coli O157:H7 were monitored in contaminated soil and farmyard manure
subjected to earthworm digestion over 21 days. Our findings lead to the conclusion
that anecic earthworms such as Lumbricus terrestris may significantly aid vertical
movement of E. coli O157 in soil, whereas epigeic earthworms such as Dendrobaena veneta significantly aid lateral movement within compost. Although the
presence of earthworms in soil and compost may aid proliferation of E. coli O157
in early stages of contamination, long-term persistence of the pathogen appears to
be unaffected.
Introduction
There is an increasing awareness that waste management
needs to be an integral part of a sustainable society,
necessitating diversion of biodegradable fractions from
landfill into alternative management processes such as
composting. This generates substantial volumes of ‘green
waste’-derived composts for commercial markets. Such
composts may include animal-derived wastes, which frequently harbour pathogenic bacteria such as Escherichia coli
O157:H7 (Hutchison et al., 2004; Nicholson et al., 2005).
Escherichia coli O157:H7 is an intestine-inhabiting bacterium associated with many severe outbreaks of disease
worldwide. While typically asymptomatic in animals, human infection may lead to haemorrhagic colitis, haemolytic
uraemic syndrome or even death (Chart, 2000). Although
E. coli O157:H7 is harbored by a range of different animals,
cattle represent the main environmental reservoir. Infected
animals typically excrete 102–105 CFU of E. coli O157:H7
per gram of feces (Hutchison et al., 2004); however, recent
2006 Federation of European Microbiological Societies
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c
studies have recovered up to 108 CFU E. coli O157:H7 per
gram of feces (Besser et al., 2001; Fukushima & Seki, 2004).
The human infectious dose is very low, and ingestion of as
few as 10 cells is thought to be sufficient to cause illness
(Chart, 2000).
The high temperatures generated within thermophilic
compost (50–70 1C) destroy mesophilic organisms such as
E. coli O157:H7 (Ndegwa & Thompson, 2001; Jones &
Martin, 2003). However, the presence of mutant thermophilic strains and/or failure to maintain high temperatures
for a sufficient length of time may lead to survival and
growth of the bacterium (Droffner et al., 1995; Elorrieta
et al., 2003; Nicholson et al., 2005). In addition, the observed
long-term persistence of this organism in manure-amended
soil (Bolton et al., 1999; Jiang et al., 2002) raises concerns
about the use of compost for soil fertilization. Growing salad
vegetables in E. coli O157:H7 contaminated soil or compost
may pose a health risk as such vegetables are frequently
consumed raw, and the bacterium may survive and even
grow on their surface (Abdul-Raouf et al., 1993), or may
FEMS Microbiol Ecol 58 (2006) 54–64
55
Earthworms as vectors of E. coli O157:H7
become internalized within tissue (Solomon et al., 2002;
Jablasone et al., 2005). Furthermore, pre-harvest contamination of vegetables with E. coli O157:H7-infected compost
is known to be responsible for enterohaemorrhagic food
poisoning outbreaks (Islam et al., 2005).
Using earthworms as a substitute to thermophilic composting or as a secondary waste treatment method (vermicomposting) is becoming increasingly common as several
studies have shown increased plant growth and yield when
grown in the presence of vermicomposts (Atiyeh et al., 2000;
Arancon et al., 2004a, b; Lee et al., 2004).
Earthworms can exert a considerable influence on the
surrounding microbial community, and may promote microbial activity within soil and composts due to the faecal
material or ‘casts’ they produce, which provide a rich carbon
source (Ndegwa & Thompson, 2001; Li et al., 2002). Earthworms may also indirectly induce significant movement of
faecal indicator organisms and pathogens via mass water
flow through abandoned channels (Joergensen et al., 1998;
Artz et al., 2005). Different earthworm species inhabit
different soil regions according to whether they are ‘anecic’
(inhabiting deep soil layers, e.g. Lumbricus terrestris) or
epigeic (inhabiting surface organic layers, e.g. Dendrobaena
veneta) species (James & Hendrix, 2004; Parkinson et al.,
2004). However, it is unclear whether the behavior of earthworm species affects movement of soil bacteria. As earthworms form a central part of the biological community in
most agricultural soils, their presence may thus enhance
persistence and dissemination of pathogens such as E. coli
O157:H7 within this environment. Although a few studies
have reported significant reductions of faecal coliforms and
Salmonella ssp. during vermicomposting (Murry & Hinckley, 1992; Eastman et al., 2002), the fate and movement of
E. coli O157:H7 in compost remains unclear. Furthermore,
vermicomposting is not yet considered by the US Environmental Protection Agency as an alternative method for
pathogen reduction for ‘class A’ products (biosolids than
can be land applied without any pathogen-related restrictions at the site and can be sold bagged to the public (EPA,
1999; Tognetti et al., 2005)). As earthworms are often
commercially bred in a matrix containing cattle manure
and other waste materials, this may potentially serve as a
vector for generating large volumes of E. coli O157:H7contaminated compost.
The aim of this current study was to assess the impact of
earthworm activity on E. coli O157:H7 movement and
persistence in soil and vermicomposts.
Materials and methods
Soil, compost, and manure collection and
characterization
Soil (Eutric cambisol of the ‘Denbigh’ series, 0–15 cm,
Table 1) and earthworms (L. terrestris) were collected from
a sheep-grazed pasture at Abergwyngregyn, North Wales,
UK (53113.9 0 N, 410.9 0 W). Earthworm bedding material
(digested paper pulp and green waste (Roberts et al., 2006)
and earthworms (D. veneta) were collected from commercial
composting beds at the same site. Aged (41 month old)
cattle manure was collected from a commercial farm in
North Wales. After collection, all samples were stored in a
Table 1. Chemical and microbiological properties of soil, compost and manure. Values represent means SEM (n = 3)
Sample
Parameter
Soil
Compost
Manure
pH
Electrical conductivity (mS cm1)
Moisture content (g kg1)
Water holding capacity (g kg1)
DOC (mg g1 dry matter)
DN (mg g1 dry matter)
Total C (g kg1)
Total N (g kg1)
C-to-N ratio
1
dry matter)
NO
3 (mg g
1
NH1
(mg
g
dry matter)
4
P (mg g1 dry matter)
K (mg g1 dry matter)
Ca (mg g1 dry matter)
Na (mg g1 dry matter)
Background heterotrophic bacteria (log10 CFU g1)
Escherichia coli O157:H7 (log10 CFU g1)
5.96 0.11
0.33 0.04
249 4
361 4
0.13 0.03
0.04 0.01
31 1
3.0 0.3
10.2
o0.1
o0.1
o0.1
0.9 0.2
10.5 3.4
0.05 0.05
7.85
0.00
8.09 0.09
0.18 0.02
535 3
1103 13
0.60 0.24
0.07 0.03
181 3
8.7 0.7
20.9
o0.1
o 0.1
o 0.1
1.3 0.1
26.3 2.2
0.21 0.04
8.41
0.00
8.59 0.14
3.05 0.08
864 5
ND
15.4 7.91
1.89 1.07
299 6
14.2 1.5
21.1
o0.1
1.0 0.7
2.2 1.6
2.1 0.1
0.3 0.1
0.42 0.02
8.93
0.00
ND, not determined.
FEMS Microbiol Ecol 58 (2006) 54–64
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56
climate-controlled room (Hemsec Ltd, Kirkby, UK) at
20 1C, 70% relative humidity for the duration of the experimental period. This temperature was selected to reflect
summertime soil and compost temperatures.
Nutrients were extracted using 1 M KCl at a 1 : 5 w/v ratio
of soil, compost, and manure -to-1 M KCl. The samples were
extracted by shaking (250 r.p.m., 1 h, room temperature),
centrifuged for 10 min (14 000 g), filtered (Whatman no.
42), and the supernatant recovered for analysis. NO
3 and
NH1
4 were determined colorimetrically (Downes, 1978;
Mulvaney, 1996) with a Skalar SAN1 segmented flow
analyzer (Skalar Analytical, Breda, The Netherlands). Phosphate was measured colorimetrically (Murphy & Riley,
1962), and K, Na and Ca were measured using a Sherwood
Scientific 410 flame photometer (Sherwood Scientific, Cambridge, UK). Electrical conductivity (EC; Jenway 4010 EC
meter) and pH (Orion 410A pH meter) were determined
after a 1 : 1 volume in volume (v/v) dilution of the soil,
compost, and manure with distilled water. Moisture content
was determined by drying for 24 h at 105 1C and water
holding capacity was measured gravimetrically (Rowell,
1994). Total organic carbon and nitrogen were measured
using a CHN2000 elemental analyzer (Leco Corp., St Joseph,
MI), and dissolved organic carbon and dissolved nitrogen
were measured using a TC-TNV analyzer (Shimadzu Corp.,
Kyoto, Japan).
Background microbiology of samples
An enrichment technique was utilized to check for the
presence or absence of background E. coli O157:H7 in the
soil, compost and manure. This was achieved by placing 5 g
of each sample into 15 mL modified Tryptone Soya Broth
(mTSB; Oxoid Ltd., Basingstoke, UK), and shaking
(150 r.p.m., 6 h, 37 1C), before plating onto sorbitol MacConkey agar plates supplemented with 0.05 mg L1 cefixime
and 2.5 mg L1 potassium tellurite (CT-SMAC; Oxoid).
Plates were then incubated at 37 1C for 18 h, and examined
and scored for presence or absence of colonies with the
characteristic appearance of E. coli O157:H7. The detection
limit of the enrichment technique was 5 CFU g1 of matrix.
An estimate of background heterotrophic bacterial counts
was undertaken by shaking 5 g of each sample (200 r.p.m.,
15 min, room temperature) in 15 mL of sterile quarterstrength Ringers solution (Oxoid), followed by 4 10 s
bursts on a vortex mixer. Serial dilutions of the solutions
were subsequently plated in duplicate onto R2A agar
(Oxoid), and colonies were counted following incubation
for 48 h at 20 1C.
Preparation of boxes and cores
To mimic field bulk density (data not presented), 10 kg of
soil and 5 kg of compost (at field moisture contents) were
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A. P. Williams et al.
manually sieved to pass 5 mm, and spread evenly in wooden
boxes (550 550 150 mm) in triplicate. To assess lateral
movement of E. coli O157:H7, 22 g of L. terrestris (all
earthworm weights expressed as ‘live weights’) was added
to soil (representing measured field density, data not presented); and 500 g of D. veneta was added to compost
(representing typical vermicomposting densities (Williams,
2004)). Other boxes without the addition of worms were
also prepared and stored in triplicate under the same
conditions (controls). Vertically-held polyvinyl chloride
(PVC) cores (66 66 500 mm) were also prepared, containing 1.5 kg of soil and 5 g of earthworms (L. terrestris).
These were used to assess vertical movement of E. coli
O157:H7 by earthworms in soil. Cores without the addition
of worms were also prepared and stored in triplicate under
the same conditions (controls). All boxes and cores were left
for 72 h before commencing the experiment to allow worms
to acclimatize and distribute themselves evenly throughout
the soil or compost. To maintain the original moisture
contents of the soils in cores, an individual ‘water table’
was established around each core by placing the bottom of
the core into a plastic sleeve and topping up the water level
daily to a maximum height of 10 cm with an artificial
rainwater solution (Jones & Edwards, 1993). Soil and compost in boxes were maintained at their original weights by
daily watering to their original weights and misting the
surfaces with artificial rainwater. This watering method was
used to prevent leaching/movement of bacteria though
matrices via water.
Preparation of E. coli O157:H7 inoculum
An inoculum was prepared from a fresh overnight culture
(LB broth; Difco Ltd., Teddington, Surrey, UK; 18 h, 37 1C,
150 r.p.m.) of an environmental isolate of E. coli O157:H7
(strain #3704 (Campbell et al., 2001)). The strain has been
proven to be nontoxigenic on the basis of lack of toxin gene
expression (Campbell et al., 2001), but it (and similar
strains) still accurately reflect survival patterns of toxigenic
E. coli O157:H7 strains (Kudva et al., 1998; Bolton et al.,
1999; Ritchie et al., 2003). Cells were washed and concentrated by centrifugation as described in Avery et al. (2005).
Preparation and application of spiked manure
A 120 mL aliquot of the E. coli O157:H7 inoculum was
added to 2.3 kg of manure and thoroughly mixed to give a
final concentration of approximately 3.0 108 CFU g1
manure (to imitate a ‘worst case’ scenario where initial
contamination levels are similar to the highest naturally
encountered (Besser et al., 2001; Fukushima & Seki, 2004)).
This was determined by enumeration on CT-SMAC agar as
described previously. Spiked manure (200 g) was then
applied in a linear band at one end of each box.
FEMS Microbiol Ecol 58 (2006) 54–64
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Earthworms as vectors of E. coli O157:H7
Harvests
Harvests were performed 1, 3, 7, 14 and 21 days after
application of inoculated manure to the compost or soil. At
each harvest, 5 g of soil or compost was gathered from three
linear points in the boxes (2 5 cm and one central point at
25 cm from box edge) and cores (2 4 cm and one central
point at 6 cm from core edge) at distances of 0, 10, 20, 30
and 40 cm from the manure band (Figs 1a and b), and
placed into individual 31 mL sterile plastic bottles. Due to
their narrow dimension, harvesting of cores was destructive
(i.e. three cores per harvest selected randomly on each date
and discarded after sampling) to avoid unrealistic results
due to disturbance of soil structure.
Bottles were subsequently shaken at 200 r.p.m. for 15 min
at room temperature in 15 mL of sterile quarter-strength
Ringers solution, followed by 4 10 s bursts on a vortex
mixer. Dilutions were plated out in duplicate onto CTSMAC agar, then incubated and colonies enumerated as
described above. As CT-SMAC plate counts approached
their theoretical detection limit (20 CFU g1), enrichment
was carried out as described above to determine whether low
numbers of culturable cells were still present. Where E. coli
O157:H7 was detectable only following enrichment, the
sample was assigned an arbitrary value equal to half of the
detection limit of plate counts (i.e. 10 CFU g1). Boxes and
cores were re-randomized within the growth room at each
harvest.
Determination of E. coli O157:H7 numbers on
and within earthworm tissue
Due to time constraints, this experiment was performed
using only one species of earthworm. Five grams of D. veneta
were starved for 12 h in Petri dishes, and then fed 5 g of
manure spiked with E. coli O157:H7 inoculum (prepared as
described previously) to a final concentration of approximately 5.0 105 CFU g1 manure. This was performed in
triplicate. After overnight feeding (12 h), worms were subsequently transferred to clean Petri dishes containing damp
filter paper until all the cast contained within the earthworm
had been excreted (4 h, adapted from Toyota & Kimura,
2000), and their intestines were empty (as verified by visual
inspection under an illuminated microscope). Subsequently,
excess cast was removed from the surface of the worms with
sterile tweezers. Casts and washings were collected, weighed
and placed in sterile plastic bottles containing 15 mL of
sterile quarter-strength Ringers solution as above. To determine the number of E. coli O157:H7 bacteria on their
surface, the worms themselves were placed in separate
plastic bottles containing 15 mL of sterile quarter-strength
Ringers solution. Thereafter, all bottles were shaken (250
r.p.m., 20 min, room temperature), and the solutions diluted, plated, incubated and enumerated on CT-SMAC agar
as described previously. To determine the number of
E. coli O157:H7 bacteria held within the worms’ intestines,
the same worms were ground in a sterile pestle and mortar,
shaken in 15 mL of sterile quarter-strength Ringers (200
r.p.m., 15 min, room temperature), and the colonies plated
and enumerated as described previously.
Total microbial activity
Microbial activity can be evaluated by studying the rate at
which simple sugars, such as glucose, are mineralized by the
microbial population (Jones et al., 2004). The total microbial
activity of soil, compost and manure, along with ‘wormcasted’
soil and compost was determined according to Palomo et al.
(2006). Briefly, 500 mL of 14C-labeled glucose (50 mM) was
added to 5 g of field-moist soil, compost and manure
contained in a 60 mL polypropylene tube; and 14CO2 evolution measured of over a 360-h period using a Wallac 1409
Liquid Scintillation Counter (Wallac Oy, Turke, Finland).
Box containng soil or compost
(a)
Manure band
0
15
0 cm
10 cm
550 mm
20 cm
30 cm
40 cm
Manure band
m
Sampling points
Sampling points
Soil core
m
0 cm
10 cm
20 cm
500 mm
30 cm
40 cm
66
Fig. 1. Experimental design of vermicomposting and soil boxes (a) and soil cores (b).
FEMS Microbiol Ecol 58 (2006) 54–64
(b)
550 mm
mm
Correspondingly, 5 g of the manure was applied to the
surface of the soil cores. This gave triplicates of six treatment
combinations as follows: soil boxes L. terrestris earthworms; compost boxes D. veneta earthworms; soil
cores L. terrestris earthworms.
66 mm
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58
A. P. Williams et al.
Plate count data from E. coli O157:H7 were log10 transformed and analyzed using a multi-factorial analysis of
variance (ANOVA) on GENSTAT 7 (VSN International Ltd.,
Hemel Hempstead, UK), with significant differences between treatments identified using Fisher’s LSD (least significant difference) test. To estimate glucose half-life (t1/2) in
soil, a double first-order exponential decay equation was
fitted by a least squares optimization routine to the glucose
mineralization data (Kemmitt et al., 2006) using SIGMAPLOT
8.0 (Systat Software UK Ltd., London, UK):
y ¼ Yr expðAtÞ þYb expðBtÞ
where y is the amount of 14C remaining in the soil, t is time,
Yr and Yb represent the amount of 14C-glucose partitioned
into microbial respiration and biomass production, respectively, and A and B represent the rate constants for these two
components. Based upon the assumption that the time
dependent mineralization of glucose matches their removal
from soil solution (Jones et al., 2004), the t1/2 of the soil
solution glucose pool can thus be defined as t1/2 = ln 2/A.
Results
Characterization of soil, compost and manure
The chemical and microbiological characteristics of materials before earthworm digestion are summarized in Table 1.
The pH of the soil used was slightly acidic (pH 5.96), and the
original moisture content was relatively high, at 68.9% of its
water holding capacity. Even though it possessed only low
levels of nitrogenous compounds, the C-to-N ratio was 10.2.
The soil harboured high numbers of indigenous bacteria
(7.85 log10 CFU g1 soil); however, enrichment yielded no
E. coli O157:H7 prior to inoculation. Compost and manure
were moderately basic (pH 8.09 and 8.59, respectively). The
moisture content of compost was moderate, at 48.5% of its
water holding capacity. Although their C-to-N ratios were
similar, manure possessed higher levels of both soluble C
and N compounds, the latter mostly in the form of NH1
4
(Table 1). Available phosphate values were much higher in
manure, whereas K and Na levels were similar for both
substrates. Ca levels were notably higher in compost. Background heterotrophic bacteria counts were somewhat greater in cattle manure than in compost (8.93 and 8.41 log10
CFU g1, respectively). No E. coli O157:H7 was detected in
either compost or manure prior to inoculation.
in numbers in the initial 24 h, the rate of decline reduced
considerably (mean log10 CFU g1 compost SEM: day 21,
0.49 0.21) (Fig. 3, Table 3). Horizontal movement of
E. coli O157:H7 was evident at early stages in the experiment, with the pathogen being detected 30 cm and 40 cm
away from the inoculation area from day 7 onwards (mean
log10 CFU g1 compost SEM: day 21, 40 cm, 0.95 0.47)
(Fig. 3, Table 2). By the end of experimental period, E. coli
O157:H7 was detected throughout the experimental worm
beds but had reduced by approximately 5 log10 units.
Although both followed a notably similar survival pattern,
the number of E. coli O157:H7 recovered was consistently
higher in vermicomposts than in control compost (mean
log10 CFU g1 compost SEM: 1.79 0.35 and 0.87 0.19,
respectively) (Figs 2 and 3, Table 2). However, no lateral
movement was detected in control boxes containing no
earthworms, with all E. coli O157:H7 cells recovered at the
inoculation point.
Soil
Overall numbers of E. coli O157:H7 declined considerably
over the course of the experiment in boxes containing
earthworms; however, low numbers (mean log10 CFU g1
soil SEM: 0.08 0.07) were still recovered at the last
harvest (21 days post soil inoculation; data not presented).
Whereas a small number of E. coli O157:H7 cells (mean log10
CFU g1 soil SEM: 0.45 0.22) were recovered 20 cm
laterally away from the point of inoculation at the first
harvest, overall lateral movement was limited; with the
majority of the E. coli O157:H7 recovered at the inoculation
point. Moreover, numbers declined so that E. coli O157:H7
could only be detected at one point in the centre of the
inoculation zone and 10 cm beyond that by the end of the
E. coli O157:H7 (log10 CFU g–1 soil/compost)
Data analysis
10
8
6
4
2
0
0
Survival and lateral movement of E. coli O157:H7
Compost
Overall, the numbers of E. coli O157:H7 declined over the
course of the experiment; however, following the sharp drop
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5
10
15
20
Days
Fig. 2. Mean survival of Escherichia coli O157:H7 over 21 days in soil
cores including (––) and excluding (––) Lumbricus terrestris; and in
compost boxes including (––,––), and excluding (––.––) Dendrobaena
veneta. Curves are representative of mean log10 (y11) E. coli
O157 g1 SEM of three replicate cores (soil), and boxes (compost).
FEMS Microbiol Ecol 58 (2006) 54–64
59
Earthworms as vectors of E. coli O157:H7
Distance from E. coli O157:H7
inoculation (cm)
0
6 log 10 CFU / g compost
10
5 log 10 CFU / g compost
20
4 log 10 CFU / g compost
30
3 log 10 CFU / g compost
(a)
(b)
(c)
2 log 10 CFU / g compost
40
Distance from E. coli O157:H7
inoculation (cm)
0
1 log 10 CFU / g compost
0 log 10 CFU / g compost
10
20
30
(d)
(e)
(f)
40
10
20
30
40
10
20
30
Sampling point (cm)
40
10
20
30
40
Fig. 3. Lateral movement of Escherichia coli O157:H7 by the earthworm Dendrobaena veneta in actively vermicomposting cattle manure (panels A–C)
in comparison with control manure containing no earthworms (panels D–F). Values represent mean log10 CFU g1 compost (n = 9).
experiment. No lateral movement of E. coli O157:H7 was
detected in control boxes.
Survival and vertical movement of E. coli
O157:H7 in soil
Numbers of the pathogen increased from day 1 to day 7
(mean log10 CFU g1 soil SEM: day 1, 3.78 1.14, day 7,
5.46 0.17), but decreased thereafter towards the last harvest (mean log10 CFU g1 soil SEM: day 21, 0.49 0.30;
Fig. 6). Escherichia coli O157:H7 movement was much more
pronounced in soil cores than in boxes, with the bacterium
being recovered at all sampling points (0–40 cm from
inoculation point) on day 1, 3 and 7 (Fig. 4, Table 3).
Furthermore, numbers were markedly similar at all sampling points at day 3 and 7, varying by only approximately
1.5 log10 CFU E. coli O157:H7 g1 soil. The absence of E. coli
O157:H7 at distances greater than 10 cm from the inoculation point towards the latter stages of the experiment
coincided with the overall decrease in numbers observed at
other sampling points (Table 3). Following a sharp drop in
the first 24 h post-inoculation, the mean number recovered
from control cores gradually decreased at all subsequent
harvests (Fig. 4, Table 3). Escherichia coli O157:H7 numbers
in control cores varied by only 1.0 log10 CFU units over the
whole experimental period, in contrast to 5.0 log10 CFU
units in soil cores with earthworms (Fig. 4, Table 3). Over
the course of the experiment, E. coli O157:H7 numbers were
Table 2. Statistical comparisons of Escherichia coli O157:H7 numbers (log10 CFU g1) in earthworm-digested compost (bold font) with control
(earthworm-undigested) compost
Distance from Escherichia coli O157:H7 inoculation (cm)
0
Day
1
3
7
14
21
10
Mean log10
CFU g1
5.87 0.04
6.29 0.12
3.96 0.48
3.86 0.07
1.04 0.54
Sig.
6.92 0.03
6.03 0.03
4.63 0.43
2.51 0.06
2.50 0.06
NS
NS
20
Mean log10
CFU g1
4.36 0.27
1.61 0.96
2.74 0.38
1.51 0.49
0.32 0.16
Sig.
0.00
0.00
0.00
0.00
0.00
NS
NS
30
Mean log10
CFU g1
1.76 0.40
3.12 0.40
2.04 0.32
0.31 0.31
0.00
Sig.
0.00
0.00
0.00
0.00
0.00
NS
NS
40
Mean log10
CFU g1
0.00
0.00
0.85 0.16
0.00
0.14 0.14
Sig.
0.00
0.00
0.00
0.00
0.00
NS
NS
NS
NS
Mean log10
CFU g1
0.00
0.00
1.84 0.49
2.24 0.23
0.94 0.46
Sig.
0.00
0.00
0.00
0.00
0.00
NS
NS
NS
t-test, where P o 0.05; P o 0.01; P o 0.001 (n = 9).
Sig., significance level; NS, not significant.
FEMS Microbiol Ecol 58 (2006) 54–64
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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60
A. P. Williams et al.
Table 3. Statistical comparisons of Escherichia coli O157:H7 numbers (log10 CFU g1) in earthworm-digested soil (bold font) with control (earthwormundigested) soil
Distance from Escherichia coli O157:H7 inoculation (cm)
0
10
20
Mean log10
Sig. CFU g1
Mean log10
Day CFU g1
6.66 0.05 6.98 0.02 1
3
7
14
21
5.87 0.07
5.61 0.18
1.75 0.88
1.25 0.64
5.94 0.00
4.77 0.02
3.12 0.01
2.90 0.02
5.85 0.17
5.31 0.25
4.78 0.15
NS 1.35 0.67
NS 1.23 0.64
NS
30
Mean log10
Sig. CFU g1
2.98 1.49
4.18 0.04
3.69 0.06
2.48 0.14
2.18 0.05
NS
NS
3.90 1.94
5.54 0.50
5.63 0.23
0.00
NS 0.00
40
Mean log10
Sig. CFU g1
0.00
0.00
1.53 0.76
0.00
0.00
NS
NS
NS
1.81 0.90
4.32 0.12
5.72 0.09
0.00
0.00
Mean log10
Sig. CFU g1
0.00
0.00
0.00
0.00
0.00
NS
Sig.
0.67 0.67 0.00 NS
4.35 0.15 0.00 5.56 0.17 0.00 NS
NS
0.00
0.00
0.00 NS
0.00 NS
Distance from E. coli O157:H7 inoculation
t-test, where P o 0.05; P o 0.01; P o 0.001 (n = 9).
Sig., significance level; NS, not significant.
0
(a)
(c)
6 log 10 CFU / g Soil
10
5 log 10 CFU / g Soil
4 log 10 CFU / g Soil
20
3 log 10 CFU / g Soil
2 log 10 CFU / g Soil
30
1 log 10 CFU / g Soil
0 log 10 CFU / g Soil
40
4
Distance from E. coli O157:H7 inoculation (cm)
(b)
6
8
4
6
8
4
6
8
0
(d)
(e)
(f)
Escherichia coli O157:H7 was detected in earthworm cast,
earthworm epidermis and from earthworm intestine following starvation (Fig. 5). Numbers of the bacterium in
excreted cast exceeded the initial inoculation concentration
(by 0.5 0.1 log10 CFU g1 manure). The greatest numbers
were recovered from earthworm cast, significantly higher
than from earthworm epidermis (P o 0.001; mean log10
CFU g1 earthworm SEM: 4.9 0.1), which in turn were
significantly higher than those recovered from the intestine
(P o 0.001; mean log10 CFU g1 earthworm SEM: 3.3 0.1) (Fig. 5).
10
Total microbial activity
20
30
40
4
6
8
4 6 8
4
Sampling point (cm)
6
8
Fig. 4. Vertical movement of Escherichia coli O157:H7 by the earthworm Lumbricus terrestris in soil (panels a, b and c) in comparison with
control soil containing no earthworms (panels d, e and f). Values
represent mean log10 CFU g1 soil (n = 9).
higher in soil cores containing earthworms than in control
cores (mean log10 CFU g1 soil SEM: 3.09 0.17 and
1.63 0.23, respectively). However, whereas numbers of
the bacterium were much higher in initial stages of the
experiment in soil containing earthworms, a drastic decline
was observed following day 7, after which fewer numbers of
the organism were recovered than from control cores (Fig. 4,
Table 3).
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
Escherichia coli O157:H7 numbers on and within
earthworm tissue
The initial rates of mineralization of glucose by indigenous
microorganisms were rapid in all substrates. In the first 48 h,
earthworms did not reduce t1/2 of 14C-glucose mineralization in substrates (Table 4); however, the final mineralization rates in substrates containing earthworms were higher
than in the controls (Fig. 6). In soil, the presence of earthworms significantly increased microbial activity and therefore the final concentration of glucose mineralized (P o
0.05). Conversely, the difference in microbial activity between earthworm-digested and -undigested compost was
not significant (P 4 0.05; Fig. 6). Two weeks after the
addition of glucose, a comparative assessment of microbial
activity showed that total 14C-glucose mineralization by
substrate microorganisms was as follows (Fig. 6): earthworm-digested compost 4 earthworm-digested soil 4compost 4 manure 4 soil.
Discussion
The reduction of human pathogens resulting from earthworm digestion has been reported previously (Eastman
FEMS Microbiol Ecol 58 (2006) 54–64
61
14CO respired
2
(%age of total 14C labelled glucose applied)
Earthworms as vectors of E. coli O157:H7
E. coli O157:H7 (log10 CFU g –1)
7
6
5
4
3
2
1
0
Cast
Epidermis
50
40
30
20
10
0
0
100
Internal tissue
200
300
400
Hours
Fig. 5. Distribution of Escherichia coli O157:H7 in the earthworm
Dendrobaena veneta after feeding on contaminated cattle manure for
12 h. The initial E. coli O157:H7 inoculation level was 5.7 log10 CFU g1
manure and is represented by the solid line. Values represent means SEM (n = 3).
Table 4. Variability of microbial mineralization of 14C-glucose expressed
as half life (t1/2) values in the initial 48 h following glucose addition
Substrate
t1/2 (h)
r2
Soil
Compost
Manure
Earthworm-digested soil
Earthworm-digested compost
23 7
30 6
14 3
40 4
61 9
0.967
0.988
0.987
0.999
0.998
r2 denotes the variability of each data point compared to that predicted
by the double-exponential decay equation.
et al., 2002; Dominguez, 2004; Edwards & Arancon, 2004).
However, several studies have shown that Gram-negative
soil bacteria can survive passage through the earthworm gut
(Thorpe et al., 1993; Hendriksen, 1995).
Digestion of organic matter by earthworms imposes
significant changes on a range of chemical, physical and
biological characteristics (Li et al., 2001; Ndegwa & Thompson, 2001). Of particular interest in this case is the reported
increase in soil microbial activity resulting from an increased availability of easily catabolized compounds expelled in earthworm casts (Tiunov & Scheu, 2000; Li et al.,
2002), competition by earthworm intestinal flora (Thorpe
et al., 1993), and the secretion of immuno-protective and
antimicrobial compounds (Cho et al., 1998; Cooper et al.,
2002; Wang et al., 2003).
Although it is known that earthworms aid movement of
microorganisms through soil (Brown, 1995; Joergensen
et al., 1998), the exact method of transportation is unclear.
In this current study, little or no movement of E. coli
O157:H7 was observed where earthworms were absent;
hence the movement observed in populated boxes or cores
can be attributed directly to earthworm activity. At present,
FEMS Microbiol Ecol 58 (2006) 54–64
60
Fig. 6. Time-dependent mineralization of 14C-labeled glucose to 14CO2
in soil (), compost (.), manure (’), Lumbricus terrestris earthwormdigested soil (), and Dendrobaena veneta earthworm-digested compost
(,) (as a % of total 14C added). The substrate concentration initially
added was 50 mM for glucose. Values represent means SEM (n = 3).
the prevalence of E. coli O157:H7 in earthworms resulting
from ingestion of infected wastes is unknown. The earthworm species in soil and compost were chosen to represent
the typical species found in each respective substrate. It was
anticipated that the direction of pathogen movement via
worms would be species-specific as earthworms species
differ in the ecological niches which they inhabit. Anecic
earthworms such as L. terrestris maintain deep vertical
burrows (James & Hendrix, 2004), whereas epigeic species
such as D. veneta inhabit surface organic layers (Parkinson
et al., 2004). In this study, E. coli O157:H7 movement by L.
terrestris was limited to a vertical plane, whereas movement
by D. veneta was observed in the horizontal plane. Although
we evaluated the carriage of bacteria on only one species,
our findings that E. coli O157:H7 may be present on the
epidermis and within the digestive tract of earthworms (Fig.
5) implies that bacterial movement may be attributed to
both worm excretion and to carriage on worm exterior;
although the relative proportions attributable to each were
not determined. Selective disinfection of their surfaces prior
to grounding worms would reduce the potential of overestimating intestinal bacteria numbers (due to ‘carry over’
from bacteria present on worm epidermis).
Along with rapid die-off of less resistant cells, the initial
drop in E. coli O157:H7 counts observed within 24 h of
inoculating the manure and application to soil and compost
may have been in part due to bacteria numbers falling to the
substrate carrying capacity (Byappanahalli et al., 2003),
competition and antagonistic effects from background bacteria, and from shock induced by a sudden change in
environmental conditions. The stabilization in numbers
observed in most treatments and the increase of 1 log10 unit
E. coli O157:H7 in soil between day 3 and day 7 (Fig. 2)
2006 Federation of European Microbiological Societies
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62
could reflect bacteria acclimatization to environmental conditions, and utilization of available food sources. It has been
shown that survival of E. coli O157:H7 varies in compost
and soils of different properties (Jones & Martin, 2003;
Franz et al., 2005). Although we chose specific temperatures
and relative humidity to reflect a particular time of year,
these factors are also likely to affect the longevity of bacteria
in different substrates. Similarly, use of a culture-based
technique can lead to an under-estimation of bacterial
numbers as E. coli O157:H7 can enter into a ‘dormant’
viable but nonculturable (VBNC) state under stress conditions such as starvation and hence be unable to grow in
nutrient-rich media. It is therefore important to note that
data obtained in the current experiment should not be
directly extrapolated to predict persistence in all soil and
vermicompost types, but rather provide a comparative study
of the influence of earthworms in these matrices under
specific conditions.
It is accepted that earthworm casts accelerate total microbial activity by increasing labile C (Tiunov & Scheu, 2000).
However, in initial stages of this study, analysis of wormcasted soil and compost revealed significantly lower
(P o 0.001) microbial activity than found in undigested
soil/compost (Table 4, Fig. 6), but significantly higher
(P o 0.001) numbers of E. coli O157:H7 (Fig. 2, Tables 2
and 3). Furthermore, where wormcasted substrates were
analyzed for microbial activity, E. coli O157:H7 numbers
were higher in those substrates with the longest glucose half
life (Table 4). The presence of earthworms in substrates and
the corresponding decrease in numbers of antagonistic
microorganisms may thus have led to an increase in
numbers of E. coli O157:H7 as observed in the initial stages
of the experiment. Protozoa are known predators of E. coli
O157:H7 in a range of environments, and several studies
have reported on the selective predation of protozoa by
earthworms (Bonkowski & Schaefer, 1997; Brown & Doube,
2004). Passage through the earthworm intestine may also
lead to further reductions in protozoa numbers (Brown &
Doube, 2004). The external structures (casts, burrows,
middens) created by earthworm activity may also produce
a ‘barrier-effect’, where microbial populations within may be
‘shielded’ due to changes in soil physical properties restricting movement of other microbes (Brown et al., 2000).
Collectively, reduced antagonism due to earthworm digestion may have facilitated the persistence of significantly
higher numbers of E. coli O157:H7 observed in wormpopulated soil and compost in the initial stages of the
current study. Nevertheless, such effects appear to have
abated with time, as the long-term persistence of E. coli
O157:H7 in soil or compost was unaffected by the presence
of earthworms. Longer-term analysis of substrate microbial
respiration suggests that activity increased in wormcasted
materials (Fig. 6), coinciding with a reduction in E. coli
2006 Federation of European Microbiological Societies
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c
A. P. Williams et al.
O157:H7 numbers; particularly in soil (Fig. 2). At the end of
the experiment, the number of E. coli O157:H7 in earthworm-digested substrates was statistically the same as in
undigested material (Fig. 2, Tables 2 and 3).
Manure from D. veneta was purged prior to exposure to
the spiked manure to increase the probability of isolating E.
coli O157:H7. It is possible that this could have led to
reduced inter-bacterial competition and thus higher pathogen numbers than would normally be encountered. Nevertheless, our results support the findings of previous studies
on the survival of Gram-negative bacteria through earthworm gut and in casts (Thorpe et al., 1993; Hendriksen,
1995). Moreover, our work suggests that earthworm digestion (Fig. 5) and presence may lead to temporarily higher
numbers of E. coli O157:H7 in some substrates, especially
soil (Fig. 2). Although the gut transit time in most earthworms is approximately 1–5 h, this may prove sufficient to
allow partial bacteria growth or for the resuscitation of VBNC
bacteria; especially where worms feed upon organic-rich
materials (Brown & Doube, 2004) as in the current study.
Earthworms are known to synthesize and secrete a variety
of immunoprotective proteins which mediate lytic reactions
against several microorganisms (Cooper et al., 2002). In
addition, one antimicrobial peptide, Lumbricin I, isolated
from adult Lumbricus rubellus, has been reported to display
antimicrobial activity against one serotype of E. coli (Cho
et al., 1998). Our work suggests that this is not effective
against E. coli O157:H7 over short time periods and at high
contamination levels, or that these antimicrobial peptides
are present in insufficient amounts to be effective against
elevated numbers/densities of E. coli O157:H7. A recent
study identified a similar antimicrobial peptide produced on
the epidermal layer of the earthworm Pheretima tschiliensis
(Wang et al., 2003). In this study, numbers of E. coli
O157:H7 were somewhat reduced on earthworm epidermis
relative to numbers in the initial inoculum (Fig. 5), and the
presence of a similar peptide might explain this; however,
this phenomenon has not been reported for D. veneta to date.
We conclude that L. terrestris are not significant vectors
for lateral movement of E. coli O157:H7 in soil; however,
these earthworms may significantly aid vertical movement.
Litter-dwelling earthworms such as D. veneta can significantly aid lateral movement of E. coli O157:H7 within
compost. Our results imply that whilst long-term persistence of E. coli O157:H7 in soil and compost may be
unaffected by the presence of earthworms, digestion from
worms may aid proliferation of the pathogen during initial
stages of soil or compost contamination.
Acknowledgements
We are grateful to the BBSRC Agri-Food and Organic
Resource Management Ltd, Canterbury, Kent, UK, for
FEMS Microbiol Ecol 58 (2006) 54–64
63
Earthworms as vectors of E. coli O157:H7
funding this project. We also wish to thank H. T. Williams
for supplying waste, and Jim Frith for preparing the boxes.
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